Bactericidal, protistocidal and nematodicidal properties of mixtures of alkyldimethylbenzyl ammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and formaldehyde Текст научной статьи по специальности «Биологические науки»

Regulatory Mechanisms

in biosystems

Regulatory Mechanisms

in Biosystems

ISSN 2519-8521 (Print) ISSN 2520-2588 (Online) Regul. Mech. Biosyst., 9(4), 540-545 doi: 10.15421/021881

Bactericidal, protistocidal and nematodicidal properties of mixtures of alkyldimethylbenzyl ammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and formaldehyde

V. V. Zazharskyi*, P. Davydenko*, O. Kulishenko*, V. Chumak*, A. Kryvaya*, I. A. Biben*, N. M. Tishkina*, I. Borovik*, O. O. Boyko*, V. V. Brygadyrenko*, **

*Dnipro State Agrarian and Economic University, Dnipro, Ukraine **Oles Honchar Dnipro National University, Dnipro, Ukraine

Article info

Received 12.10.2018 Received in revised form

14.11.2018 Accepted 17.11.2018

Dnipro State Agrarian and Economic University, SergiyEfremov st., 25, Dnipro, 49000, Ukraine. Tel.: +38-056-713-51-74. E-mail:

boikoalexandra1982@gmail.com

Oles Honchar Dnipro National University, Gagarin av., 72, Dnipro, 49010, Ukraine. Tel.: +38-050-93-90-788. E-mail: brigad@ua.fm

Zazharskyi, V. V., Davydenko, P., Kulishenko, O., Chumak, V., Kryvaya, A., Biben, I. A., Tishkina, N. M., Borovik, I., Boyko, O. O., & Brygadyrenko, V. V. (2018). Bactericidal, protistocidal and nematodicidal properties of mixtures of alkyldimethylbenzyl ammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and formaldehyde. Regulatory Mechanisms in Biosystems, 9(4), 540-545. dou-10.15421/021881

We conducted a comparative analysis of the impact of disinfecting preparations on the cryogenic stains of microorganisms, and also on Haemonchus contortus (Rudolphi 1803), invasive larvae of the ruminants. To test the preparations for disinfection, we used laboratory analyses with methods of biotesting, particularly with the use of Paramecium caudatum Her., Tetrahymena pyriformis Ehrenberg. We researched mixtures of substances: alkylbenzyldimethylammonium chloride (C24H42IN, BAK, mixture of homologues alkylbenzyldimethylammonium chloride and with n-C12H25, n-C14H29 and n-C16H33), didecyldimethylammonium Chloride (DDAC, C22H48ClN) and glutaraldehyde (C5H8O2); formaldehyde (CH2O), alkylbenzyldimethylammonium chloride and glutaraldehyde in 1% have bactericidal properties for the following cryogenic strains of microorganisms: Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, Proteus vulgaris, Serracia marcescens, Pseudomonas aeruginosa, Enterococcus faecalis and Yersinia enterocolitica. The Bacillus cereus were affected by the preparations bacteriostatically: we observed growth in the colonies in the medium with addition of 1% solution of mixture of alkylbenzyldimethylammonium chloride and didecyldimethylammonium chloride, and also 1%, 5% and 10% of solution of mixture of glutaraldehyde, formaldehyde and alkylbenzyldimethylammonium chloride. Also, these mixtures of substances have nematocidal properties. Death of 100% of L3 H. contortus after 24 hour exposure was observed with use of 1% solution of mixture of alkylbenzyldimethylammonium chloride and didecyldimethylammonium chloride, and also 5% - glutaraldehyde, formaldehyde and alkylbenzyldimethylammonium chloride. Effective disinfection measures perform a leading role in providing stable veterinary well-being of livestock and healthcare of the population. Maximum toxicity during usage of the mixtures on P. caudatum was observed for the mixture of alkylbenzyldimethylammonium chloride and didecyldimethylammonium chloride, and also for formaldehyde and glutaraldehyde. The lowest toxicity for T. pyriformis was observed with use of the mixture of glutaraldehyde, sodium dodecylsulfate (SDS) and oleum terebinthini, and also the mixture of formaldehyde and glutaraldehyde, the highest - formaldehyde and alkylbenzyldimethylammonium chloride. Thus, the most promising mixtures for use in veterinary medicine were determined to the following: alkylbenzyldimethylammonium chloride, didecyldimethylammonium chloride and glutaraldehyde, and also formaldehyde, alkylbenzyldimethylammonium chloride and glutaraldehyde.

Keywords: disinfectant; bactericidal action; toxicity; Paramecium caudatum; Tetrahymena pyriformis; Haemonchus contortus

acute toxicity of pharmaceutical substances at the stage of their screening and pre-clinical study (Zhmin'ko et al., 2006).

The results of studies using the express-method of toxicity on cilia-tes indicated that solution of benzalkonium chloride, alkylbenzyldime-thylammonium chloride ("Geocyd") in 0.03-0.50% concentrations and 1-10 min exposure exhibited no toxic effect on the culture of T. pyriformis ciliates (Kovalenko et al., 2014).

The extent of acute toxicity at endogastric introduction of LD50 "Univait" preparation to mice equaled 5200 mg/kg of the animal's body weight. According to the results of the studies, a preparation was developed, which belongs to the fourth class by the classification of chemical substances in relation to the extent of toxicity. "Univait" disinfecting preparation in 0.1-0.5% concentrations during 10 min exposure was insignificantly toxic to the cultures of T. pyriformis ciliates (Zasekin et al., 2016). For predicting toxicity of aromatic aldehydes for T. pyriformis, mathematical models are proposed, particularly the linear and nonlinear models (Ousaa et al., 2018). At the same time, there are data on the impact of aromatic aldehydes on nematode parasites of agricultural

Introduction

An obligatory component in the system of veterinary-sanitary measures for the objects of livestock farming is performance of disinfection. Prevention of diseases of infectious etiology conditioned by condition-nally-pathogenic microflora requires disruption of the epizootic chain of distribution of diseases from sources of infection. A leading role in provision of stable veterinary well-being of livestock farming and healthcare of the population is played by the conducting of effective disinfect-tion measures which also cause the least possible harm to the environment. Therefore, disinfection preparations are tested using laboratory analyses with methods of biotesting, particularly with ciliates. By toxicity for the ciliates, the substances are divided into four classes: 1 (LC over 0.001%), 2 (LC over 0.1%), 3 (LC over 1%), 4 (non-toxic) (Kotsumbas et al., 2006).

Correlation between the parameters of toxicity during comparative study of acute toxicity for the laboratory animals, ciliates 6 indicates that the ciliate T. pyriformis can be used an alternative model in predicting

animals. At the impact (24h) of 1% solution of cinnamaldehyde, there was observed death of 100% of eggs of Ascaris suum (LD50 = 2437 ± 864 mg/l) (Boyko & Brygadyrenko, 2017a). Larvae of Strongyloides ransomi, nematodes of pigs, also died over 24 hours at the impact of 0.1%o solution of benzaldehyde. LD50 for benzaldehyde - 142 ± 64 mg/l (Boyko & Brygadyrenko, 2017b). The literature contains a large amount of data on the impact of alkylbenzyldimethylammonium chloride, dide-cyldimethylammonium chloride, formaldehyde, glutaraldehyde and other certain substances on microorganisms (Braoudaki et al., 2005; Blon-deau et al., 2007; Fazlara & Ekhtelat, 2012; Vaerewijck et al., 2012; Ivancovic et al., 2013; Lasemi, 2017). Therefore, the objective of our study was to perform a comparative assessment of bactericidal, protisto-cidal and nematocidal properties of mixtures of alkylbenzyldimethylammonium chloride, didecyldimethylammonium chloride and glutaraldehyde; alkylbenzyldimethylammonium chloride, formaldehyde and glutaraldehyde; sodium dodecyl sulfate (SDS), oleum terebinthini and glutaraldehyde, and also formaldehyde and glutaraldehyde.

Materials and methods

The research was conducted in the laboratories of the departments of Epizootology and Infectious Diseases of Animals, Physiology and Biochemistry of Agricultural Animals, Parasitology and Veterinary-Sanitary Examination of the Faculty of Veterinary Medicine of Dnipro National Agrarian-Economic University, and also in the Bacteriological Department of Dnipro Regional National Laboratory of Veterinary Medicine in 2017-2018.

Bacteria. The cultures of microorganisms of standardized strains (Table 2), cultivated on a dense growth medium over 18-24 hours were washed out with sterile isotonic solution of sodium chloride at temperature of 37 ± 2 °C. The weighed microbial amounts were processed to 5 ■ 108 CFU/ml of McFarland turbidity standard for optical standartisa-tion of bacteria using a Dilushaker III Digital densitometer, France. The solutions of disinfectants in the working concentration (0.9 ml) were poured into sterile test tubes. To the test tubes with disinfectant solutions (1, 5, 10, 25%), 0.1 ml of weighed microbial amounts were added, mixed, and then the tubes were shaken for a few seconds (Table 1).

Then, 0.5 ml of solution of the neutralizer was added (Tvin-80 -3%, saponin - 3%, histidine - 0.1%, cysteine - 0.1%) and the tubes were shaken. The inoculations were made on to a specific differential-diagnostic medium by 0.1 ml of the mixture, and the cups with inoculated cultures were put in a thermostat for 24 hours. The methods are descrybed in detail in the articles by Zazharskyi et al. (2018a, 2018b).

The incubation was performed in accordance with the passport for the growth media. After the time necessary for the cultivation of the studied microorganisms, we assessed the number of the microorganisms that grew in the Petri dish. Distinctive typical colonies were re-inoculated to beef-extract agar and incubated for 24 hours at 37 °C. The cultivated colonies underwent microscopy. If necessary, an additional identification of microorganisms was conducted in accordance with EN ISO 11133: 2014, IDT (Table 2).

Ciliates. Comparative analysis of the impact of disinfecting preparations on criogenic strains of microorganisms was performed in accordance with the generally accepted methods. The cultivation of P. cau-datum and T. pyriformis ciliates was done in lactic media. The culture was maintained at room temperature (18-20 t). For the biotesting, we used a 24-hour culture which was in the phase of exponential (active) growth. To conduct the toxicological study, we prepared a series of dissolved preparations (Table 1): 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% and 0.000001%.

In 5 micro-aquarium cavities 20 ^l of the medium with ciliates (1020 individuals) were put. Then 20 ^l of aquatic solution of the studied preparations of different concentrations was added to each cavity and the number of cells in each aquarium was assessed. After 1 hour exposure, we again assessed the number of P. caudatum in each cavity of the aquarium and determined the percentage of their survival. During use of T. pyriformis culture, due to the small sizes of the cells and the impossibility of counting them precisely, the assessment of the biotest results was performed in relation to death of ciliates and the pattern of changes

in movement. For most substances, we determined almost complete similarity in the decline angle of the straight line of lethality for ciliates and laboratory animals. This allows us to extrapolate the results of studies on protozoans to animals and humans. The values of LD50 for all studied substances, obtained using the method of expressive biotesting are within confidence intervals for LD50 values obtained experimentally (Miyoshi et al., 2003; Venkateswara et al., 2007).

Table 1

Mixtures of substances used

Name of preparation

Mixture composition

Formula

Amount of substance, g/kg

alkylbenzyldimethylam monium chloride (BAK)

"Aldovet

FF'

didecyldimethyl ammonium chloride (DDAC)

glutaraldehyde

alkylbenzyldimethyl-ammonium chloride

"Aldovet 2 super plus"

DZPT-2 disinfectant preparation against tubercu-losis-2

FAG glutaric formaldehyde

formaldehyde glutaraldehyde

sodium dodecyl sulfate (SDS)

oleum terebinthini

glutaraldehyde

formaldehyde

glutaraldehyde

170.6

78.0

107.25

25.0

168.0

225.0

0 0

250.0

'0' N0"Na+

50.0

250.0

200.0

200.0

Nematoda. The larvae of nematodes in feces of ruminants were found using the Baermann test (Zajac et al., 2011). Then, 1 ml of the studied mixtures of the substances in different concentrations (1%, 5%, 10%, 25%) was added to each culture of H. contortus nematode larvae (in five times replication). The experimental exposure equaled 24 hours. We determined the number of vital and dead larvae. The methods are described in detail in articles by Boyko & Brygadirenko (2018a, 2018b).

Statistical analysis. The extrapolation of the data on acute toxicity of the studied substances, obtained for T. pyriformis, to animals was implemented in accordance with the recommended methods of express biotesting. For this purpose, effective dose of a certain substance, obtained in the experiment in determining acute toxicity, was expressed as probit which was placed in the graph of the lethality line of T. pyriformis ciliates and LC50 value was calculated. The results are satisfactory if LC50 value obtained using the method of express biotesting is within the confidence interval (error). Value of LC50 for ciliates was determined using probit-analysis of lethality curves. Probit-analysis is recommend-

3

4

ded by OECD Guidelines for the Testing of Chemicals for assessment of harmful impact of different toxicants. The statistical analysis of the results with H. contortus was performed through a set of Statistica 8.0 (StatSoft Inc., USA), the figures show the median, 25% and 75% quar-tiles, minimum and maximum values. LD50 (%) was calculated as mean ± standard deviation (x ± SD).

Results

The mixtures we studied - alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde, and also alkyl-

Table 2

Studied growth media

benzyldimethylammonium chloride, formaldehyde, and glutaraldehyde -demonstrated bactericidal properties even in 1% concentration against cryogenic strains of the following microorganisms: S. aureus, S. typhi-murium, E. coli, L. monocytogenes, P. vulgaris, S. marcencens, P. aeruginosa, E. faecalis and Y. entemcolitica. The mixtures of these substances demonstrated a bacteriostatic effect on B. cereus microorganisms: growth was observed in the colonies with addition of 1% of solution of mixture of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, and glutaraldehyde, and also 1%, 5% and 10% solutions of mixture of alkylbenzyldimethylammonium chloride, formaldehyde, glutaraldehyde (Table 3).

Stains of microorganisms

Growth medium, HiMedia Laboratories Pvt. Limited (India)

No of medium

base

reason for study

Staphylococcus

aureus ATCC 25923

Salmonella typhimurium 144

Bacillus cereus ATCC 10702

Escherichia coli

(F 50) ATCC 25922

Listeria monocytogenes ATCC19112

Proteus vulgaris HX19 222

Seratia marcescens 1

Pseudomonas

aeruginosa ATCC 2853(F)

Enterococcus

faecalis ATCC 19433

Yersinia enterocolitica

M043-500G Baird Parker agar base

xylose lysine M031-500G deoxycholate agar (XLD agar)

M833-500G

Bacillus cereus agar base

Baird Parker agar base, 500 g (REF 2009/03709) {ISO 6887:2003}

xylose lysine deoxycholate agar

(XLD agar), 500 g (REF 2009/03709) {ISO 6887:2003}

Bacillus cereus agar base, 500 g (REF 2009/03709) {ISO 6887:2003}

M065A

deoxycholate citrate agar (as per B.P.)

M1075-500G endo agar, modified

for selection and assessment of coagulase-positive Staphylococcus in food products and other examined material; FD046 egg yolk tellurite emulsion (100 ml/vial) / yolk emulsion with tellurite; FD069 B P sulpha supplement / additive with sulfamethazine for Baird Parker medium

for selection and assessment of Salmonella typhi and other Salmonella

FD003 polymyxin B selective supplement / FD045 egg yolk emulsion (100 ml/vial); for selection and count of colonies of anthracoid Bacillus; FD003 polymyxin B selective supplement; FD045 egg yolk emulsion (100 ml/vial)

deoxycholate citrate agar for selection of pathogens of intestinal infections

M1064-500G

Listeria identification agar base (PALCAM)

MacConkey agar w/o M082-500G CV, NaCl w/sodium taurocholate 0.5%

M001-500G nutrient agar

M085-500G Pseudomonas agar base

M001-500G nutrient agar M1075-500G endo agar, modified

M001-500G nutrient agar M1075-500G endo agar, modified

endo agar, modified, 500 g (REF 2009/03709) Listeria identification agar (base)

(PALCAM), 500 g (REF 2009/03709) {ISO 6887:2003}

MacConkey agar without crystal violet, NaCl, with 0,5%

taurocholic acid sodium, 500 g (REF 2009/03709)

nutrient agar, 500 g (REF 2009/03709) {ISO 6579:2002} basis for the agar for Pseudomonas, 500 g (REF 2009/03709) nutrient agar, 500 g (REF 2009/03709) {ISO 6579:2002} endo agar, modified, 500 g (REF 2009/03709) nutrient agar, 500 g (REF 2009/03709) {ISO 6579:2002} endo agar, modified, 500 g (REF 2009/03709)

for identification and selection of coliform bacteria of the intestinal group

for selection and identification of Listeria; FD061 Listeria selective supplement (PALCAM)

this agar is prepared in accordance with the requirements for clinical microbiology; on this differential medium, swarming of most strains of Proteus is inhibited, which significantly facilitates the selection of intestinal bacteria; along with opportunistic gram-positive bacteria, a large number of Proteus can be maintained in it; enterococci in it form small reddish colonies

is used as the main medium for cultivating not very fastidious microorganisms or for preparing special media (after 10% of blood or other biological fluid)

is recommended with additives for selection of Pseudomonas; recommended by the International Committee (ISO); FD029 cetrinix supplement / cetrinix additive for Pseudomonas is used as the main medium for cultivating not very fastidious microorganisms or for preparing special media (after 10% of blood or other biological fluid)

for determining and selecting coliform and other bacteria of the intestinal group

is used as the main medium for cultivating not very fastidious

microorganisms or for preparing special media

(after adding 10% of blood or other biological fluid)

for identification and selection of the coliform and other bacteria

of the intestinal group_

No negative impact on the mobility of T. pyriformis was demonstrated by the mixtures of sodium dodecyl sulfate (SDS), essential oil, glutaraldehyde, and also formaldehyde, glutaraldehyde with 0.01%, mixtures of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and also alkylbenzyldimethylam-monium chloride, formaldehyde, glutaraldehyde with 0.0001% (Table 4). According to the results of our previous studies (Zazharskyi et al., 2018a, 2018b), the impact of 0.01 mg/l of mixture of alkylbenzyldime-thylammonium chloride, didecyldimethyl ammonium chloride, glutar-aldehyde and formaldehyde, and glutaraldehyde caused the highest death rate of ciliates - 26% and 22% respectively (Table 5). LC50 equaled 1.8 mg/l with use of the mixture of alkylbenzyldimethylammo-nium chloride, didecyldimethyl ammonium chloride, glutaraldehyde, 8.4 mg/l - formaldehyde, glutaraldehyde, 27.2 mg/l - alkylbenzyldime-thylammonium chloride, formaldehyde, glutaraldehyde, 53.4 mg/l -sodium dodecyl sulfate, essential oil, and glutaraldehyde. In the series of experiments on ciliates, the death of different numbers of them was ob-

served in interval 0.001-10 mg/l with use of the mixture of alkylbenzyl-dimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and formaldehyde, glutaraldehyde, 0.001-100 mg/l -alkylbenzyldimethylammonium chloride, formaldehyde, glutaraldehyde, 0.1-100 mg/l for sodium dodecyl sulfate, essential oil, glutaraldehyde.

The greatest impact on the vitality of nematode larvae in the environment was demonstrated by alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride and glutaraldehyde. 100% death rate of H. contortus, nematode larvae of ruminants was observed with use of 1% solution of this mixture. Nematocidal effect was exhibited by the mixture of alkylbenzyldimethylammonium chloride, formaldehyde and glutaraldehyde: nematode larvae of the studied species died at 5% concentration. Mixtures of sodium dodecyl sulfate, oleum tere-binthini, glutaraldehyde, and also formaldehyde and glutaraldehyde were the least efficient against invasive larvae of H. contortus. 100% death rate of L3 larvae was observed only when 25% solution of mixtures of these substances was used (Fig. 2).

Table 3

Influence of the studied mixtures on cryogenic strains of microorganisms (n = 5)

Stains of microorganisms

Alkylbenzyldimelhylammonium chloride, didecyldimelhyl ammonium chloride, glutaraldehyde, %

Alkylbenzyldimelhyl-ammonium chloride, formaldehyde, glutaraldehyde, %

Sodium dodecyl sulfate, essential oil, glutaraldehyde, %

Formaldehyde, glutaraldehyde, %

1 5 10 25 1 5 10 25 1 5 10 25 1 5 10 25

S. aureus ATCC 25923 - - - - - - - - + - - - - - - -

S. typhimurium 144 - - - - - - - - + + - - - - - -

B. cereus ATCC 10702 + - - -+++++ - +++ +++ ++ ++ +++ +++ ++ ++

E. coli (F 50) ATCC 25922 - - - - - - - - + - - - - - - -

L. monocytogenes ATCC + - + -

19112

P. vulgaris HX19 222 ------- - + - - - - - - -

S.marcescens 1

P. aeruginosa ATCC 2853(F) - - - - - - - - ++ ++ - - ++ ++ + -

E. faecalis ATCC 19433 - - - - - - - - ++ + - - ++ ++ + -

Y. enterocolitica - - - - - - - - + - - - - - - -

Note: "—" - no growth in colonies, "+" - one colony, "++" - two colonies, "+++" - three colonies.

Table 4

Influence of studied substances on T. pyriformis (n = 5)

Concentration, Exposure, ■ % hour Types of mixtures

alkylbenzyldimethylammonium chloride, alkylbenzyldimethylammonium chloride, sodium dodecyl sulfate, formaldehyde,

didecyldimethyl ammonium chloride, glutaraldehyde formaldehyde, glutaraldehyde essential oil, glutaraldehyde glutaraldehyde

01 1 24 1 1.0 х 10-2 24 i1 ±2 -1 =t4 + ±4 +

1.0 х 10-3 24 ±5 + ±6 + + + +

1.0 х 10-4 1 24 + + ±7 + + + + +

1.0 х 10-5 24 + + + + + + + +

1.0 х 10-6 24 + + + +

+ + + +

Note: "—" - no growth (death), "±" - movement slowed, "+" - active movement; 1 - after the addition, movement intensifies, the direction changes, after 60 min - single moving individuals, movement slowed; 2 - restoration of movement, decrease in density of the culture, movement slowed; 3 - restoration of movement, decrease in density of the culture, movement slowed; 4 - insignificant decrease in density; 5 - slowed movement; 6 - rotation, slowed movement, decrease in density; 7 - slowed movement, insignificant decrease in density (Zhmin'ko et al., 2006).

Table 5

Influence of the studied substances on P. caudatum (% dead ciliates; n = 5)

Concentration of mixtures in the sample, mg/l

Mixture compound

0.01

0.1

1

10

100

control experiment control experiment control experiment control experiment control experiment

Alkylbenzyldimethylammonium chloride, didecyldimethyl 0 26 0 42 0 66 0 100 0 100

ammonium chloride, glutaraldehyde

Alkylbenzyldimethylammonium chloride, formaldehyde, 0 12 0 23 0 33 0 65 0 100

glutaraldehyde

Sodium dodecyl sulfate, oleum terebinthini, glutaraldehyde 0 0 0 12 0 12 0 7 0 100

Formaldehyde, glutaraldehyde 0 22 0 21 0 40 0 100 0 100

During usage of mixture of sodium dodecyl sulfate, oleum terebin-thini and glutaraldehyde in 5% concentration, the vitality of nematode larvae was observed on average to be 25%, in 10% concentration it was 8% of individuals. When the mixture of formaldehyde, glutaraldehyde was used in 1%, 5% and 10% concentration, on average 1-10% of individuals survived. LD50 for mixture of sodium dodecyl sulfate, essential oil and glutaraldehyde equals 2.3 ± 0.8%, for formaldehyde, glutaraldehyde it was 0.45 ± 0.16%.

Discussion

The series of studies Takashi & Kei-Ichiro (2007) proved the bactericidal effect of didecyldimethyl ammonium chloride in minimum inhibiting concentration 1.3 mg/l against E. coli. The studies by Shirron et al. (2009) allow us to state that didecyldimethyl ammonium chloride causes a bactericidal effect against S. typhimurium. Walsh et al. (2003) reported that didecyldimethyl ammonium chloride has a bactericidal effect on E. coli, S. aureus, P. aeruginosa and L. monocytogenes. According to Ioannou et al. (2007), alkylbenzyldimethylammonium chloride and dide-

cyldimethyl ammonium chloride are at the same time membrane-active agents with subtly different mechanisms of action, which reflect the previous interaction with S. aureus.

The studies by Lasemi et al. (2017) demonstrated the impact of 2% solution of glutaraldehyde on the spores of B. subtilis. The results showed that 102 colonies were present on the 10th minute, 18.6 ± 3.4 on the 15th minute, 6.2 ± 1.4 on the 20th minute, 2.1 ± 0.8 on the 25th minute and no colonies after 30 minutes. Over the first 10 minutes, more colonies were observed, after 15-20 minutes this number significantly reduced. After 30 minutes, growth of the colonies completely stopped. 2% density of glutaraldehyde over 30 minutes was sufficient for eliminating the spores ofB. subtilis. The data by Simoes et al. (2008) indicate that sodium dodecyl sulfate has an antimicrobal effect on the biomembranes of P. fluorescens. In their studies, Chen et al. (2016) mention that pathogenic strains of E. coli, P. aeruginosa and K. pneumoniae have a FrmRAB regulator, and can be used for eliminating endogenous and exogenous Formaldehyde.

Vaerewijck et al. (2012) determined that alkylbenzyldimethylammonium chloride and sodium hypochlorite in concentration of active

chlorine of 50 mg/l can inactivate Acanthamoeba and two species of Tetrahymena spp. in 15 minutes. The series of studies by Ivancovic et al. (2013) proved the lethal effect of alkylbenzyldimethylammomum chloride on Paramecium caudatum. The studies by Blondeau et al. (2007) allow us to state that alkylbenzyldimethylammonium chloride in a combination with gatifloxacin and moxifloxacinin in concentration of 0.0080.125 mg/l exhibited bactericidal effect against polyresistent S. aureus. The studies by Braoudaki et al. (2005) demonstrated that alkylbenzyldime-thylammonium chloride in combination with erythromycin has a bactericidal effect against S. typhimurium. Fazlara & Ekhtelat (2012) described the lethal influence of alkylbenzyldimethylammonium chloride on B. cereus. In their studies, Carmen Velazquez et al. (2009) also mentioned that alkyl-benzyldimethylammonium chloride has a bactericidal effect against E. coli. The studies by Bridier et al. (2011) allow us to state that alkylbenzyldimethylammonium chloride has a bactericidal effect on E. faecalis.

100 90 80 70 60 50 40 30 20 10 0

a

100 90 80 70 60 50 40 30 20 10 0

b

Fig. 2. Influence of the studied mixtures on vitality of nematode

larvae of H. contortus: a - sodium dodecyl sulfate, oleum terebinthini, glutaraldehyde, b - formaldehyde, glutaraldehyde

Ibusquiza P. Saa et al. (2011) found resistance of Listeria monocytogenes to alkylbenzyldimethylammonium chloride. Hattori et al. (2003) observed resistance to this substance by P. vulgaris. Tiwari et al. (2003) also proved the resistance of Serratia marcescens to this substance. By contrast, Paul et al. (2010) allow us to state that alkylbenzyldimethylammonium chloride shows no bactericidial effect against P. aeruginosa.

There are data on using mixtures of formaldehyde and glutaralde-hyde as a disinvasive preparation. The study by Palij et al. (2018) describes the effect of FAG aldehyde preparation on the eggs of nematodes of agricultural animals. It was determined that the preparation in 6.0% concentration at 24 hours exposition demonstrates a disinvasive effect against eggs of Ascaris suum, Ascaridia galli and Toxocara canis. Mixture of these aldehydes is an efficient preparation for disinfecting the premises of livestock contaminated with invasive helminths.

The highest bactericidal, protistocidal, and also nematodicidal effect were observed for use of mixtures of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride and glutaraldehyde, and also alkyldimethylbenzylammonium chloride, formaldehyde and glutaraldehyde.

Mixtures of alkylbenzyldimethylammonium chloride, formaldehyde, glutaraldehyde, and also alkylbenzyldimethylammonium chloride, di-decyldimethyl ammonium chloride, and glutaraldehyde demonstrated bactericidal properties on cryogenic strains of S. aureus, S. typhimurium, E. coli, L. monocytogenes, P. vulgaris, S. marcescens, P. aeruginosa, E. faecalis, and Y. enterocolitica, and also nematocidal properties against H. contortus, nematode larvae of ruminants. Maximum toxicity during use of the studied substances against P. caudatum was demonstrated by alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde, and also formaldehyde and glutaraldehyde. The least toxic were mixtures of sodium dodecyl sulfate, oleum terebinthini, and glutaraldehyde (14-15 times safer). The mixture alkylbenzyldimethylammonium chloride, formaldehyde and glutaraldehyde showed a moderate level of toxicity. The least toxicity for T. pyriformis was observed for the mixture of sodium dodecyl sulfate, essential oil, glutaraldehyde, and also formaldehyde and glutaraldehyde, the highest for alkylbenzyldimethylammonium chloride, formaldehyde, and glutaral-dehyde. The strongest effect on the viability of nematode larvae in the environment was shown by alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, and glutaraldehyde. 100% death rate of H. contortus, nematode larvae of ruminants, was recorded already at using 1% solution of this mixture. Nematocidal effect was observed for mixture of alkylbenzyldimethylammonium chloride, formaldehyde, and glutaraldehyde: nematode larvae of the studied species died at 5% concentration. Thus, our observations can be useful for practicing doctors of human and veterinary medicine during preparation of antiseptics, disinfecting and disinvasive preparations with predicted biocidal effect of four ammonium compounds.

References

Blondeau, J. M., Borsos, S., & Hesje, C. K. (2007). Antimicrobial efficacy of gatifloxacin and moxifloxacin with and without benzalkonium chloride compared with ciprofloxacin and levofloxacin against methicillin-resistant Staphylococcus aureus. Journal of Chemotherapy, 19(2), 146-151. Boyko, A. A., & Brygadyrenko, V. V. (2017a). Changes in the viability of the eggs of Ascaris suum under the influence of flavourings and source materials approved for use in and on foods. Biosystems Diversity, 25(2), 162-166. Boyko, A. A., & Brygadyrenko, V. V. (2017b). Changes in the viability of Strongyloides ransomi larvae (Nematoda, Rhabditida) under the influence of synthetic flavourings. Regulatory Mechanisms in Biosystems, 8(1), 36-40. Boyko, О. О., & Brygadyrenko, V. V. (2018a). The impact of certain flavourings and preser-vatives on the survivability of larvae of nematodes of Ruminantia Regulatory Mechanisms in Biosystems, 9(1), 118-123. Boyko, О. О., Gavrilina, O. G., Gavrilin, P. N., Gugosyan, Y. A., & Brygadyrenko, V. V. (2018b). Influence of formic acid on the vitality of Strongyloides papil-losus. Regulatory Mechanisms inBiosystems, 9(3), 435-439. Braoudaki, M., & Hilton, A. C. (2005). Mechanisms of resistance in Salmonella enterica adapted to erythromycin, benzalkonium chloride and triclosan. International Journal of Antimicrobial Agents, 25(1), 31-37. Bridier, A., Briandet, R., Thomas, V., & Dubois-Brissonnet, F. (2011). Comparative biocidal activity of peracetic acid, benzalkonium chloride and ortho-phthalaldehyde on 77 bacterial strains. Journal of Hospital Infection, 78(3), 208-213.

Chen, N. H., Djoko, K. Y., Veyrier, F. J., & McEwan, A. G. (2016). Formaldehyde

stress responses in bacterial pathogens. Frontiers in Microbiology, 7, 257. del Carmen Velazquez, L., Barbini, B. N., Escudero, M. E., & Estrada, C. L., de Guzman, A. M. S. (2009). Evaluation of chlorine, benzalkonium chloride and lactic acid as sanitizers for reducing Escherichia coli O157:H7 and Yersinia enterocolitica on fresh vegetables. Food Control, 20(3), 262-268. Fazlara, A., & Ekhtelat, M. (2012). The disinfectant effects of benzalkonium chloride on some important foodborne pathogens. American-Eurasian Journal of Agricultural and Environmental Sciences, 12(1), 23-29. Hattori, N., Sakakibara, T., Kajiyama, N., Igarashi, T., Maeda, M., & Murakami, S. (2003). Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride. Analytical Biochemistry, 319(2), 287-295. Ibusquiza, P. S., Herrera, J. J. R., & Cabo, M. L. (2011). Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes. Food Microbiology, 28(3), 418-425. Ioannou, C. J., Hanlon, G. W., & Denyer, S. P. (2007). Action of disinfectant quaternary ammonium compounds against Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 51(1), 296-306. Ivancovic, T., Hrenovic, J., & Matonickin-Kepcija, R. (2013). Resistance of bio-particles formed of phosphate-accumulating bacteria and zeolite to harsh

K 1% 5% 10% 25%

10% 25%

environmental conditions. The Journal of Bioadhesion and Biofilm Research, 29(6), 641-649.

Kotsumbas, I. Y., Malyk, O. G., & Paterega, I. P. (2006). Doklinichni doslidzhen-nya veterinarnih likarskih zasobiv [Preclinical studies of veterinary medicinal products]. Triada Plus, Lviv (in Ukrainian).

Kovalenko, V. L., Gnatenko, A. V., & Ponomarenko, G. V. (2012). Porivnialne vyznatchennya toksichnosti bacteritsydnyh zasobiv za pokaznykamy gostroyi toksychnosti ta alternatyvnyh metodiv [Comparative definition of toxicity of bactericidal agents on indicators of acute toxicity and alternative methods]. Problems of Zoinengineering and Veterinary Medicine, 25(2), 169-173 (in Ukrainian).

Kovalenko, V. L., Lyasota, V. P., & Balatskij, Y. O. (2014). Viznachennya tok-sichnosti dezinfikuyuchogo preparatu "Geotsid" z vikoristannyam infuzoriji Tetrachynema pyriformis [Determination of the toxicity of the disinfectant "Geocid" using the Tetrachynema pyriformis infusoria]. Problems of Zoinen-gineering and Veterinary Medicine, 29(2), 262-265 (in Ukrainian).

Lasemi, E., Kalantar, M. H., Navi, M. F., Rezae, M., Nikfar, N. H., Danial, Z., & Azizpour, R. (2017). Effects of different times of glutaraldehyde 2% on Bacillus subtilis spores (in vitro). Hospital Practices and Research, 2(4), 118-121.

Mc Cay, P. H., Ocampo-Sosa, A. A., & Fleming, G. T. (2010). Effect of subinhi-bitory concentrations of benzalkonium chloride on the competitiveness of Pseudomonas aeruginosa grown in continuous culture. Microbiology, 156(1), 30-38.

Miyoshi, N., Kawano, T., & Tanaka, M. (2003). Use of Paramecium species in bioassays for environmental risk management: Determination of LC50 values for water pollutants. Journal of Health Science, 49(6), 429-435.

Ousaa, A., Elidrissi, B., Ghamali, M., Chtita, S., Aouidate, A., Bouachrine, M., & Lakhlifi, T. (2018). Quantitative structure-toxicity relationship studies of aromatic aldehydes to Tetrahymena pyriformis based on electronic and topological descriptors. Journal of Materials and Environmental Science, 9(1), 256-266.

Palij, А P., & Sumakova, N. V. (2018). Viznachennya dezinvazijnikh vlastivostej dezzasobu "FAG" [Determination of disinfesive properties of disinfection "FAG"]. Veterinary Biotechnology, 32(2), 405-412.

Shirron, N., Kisluk, G., Zelikovich, Y., Eivin, I., Shimoni, E., & Yaron, S. (2009). A comparative study assaying commonly used sanitizers for antimicrobial activity against indicator bacteria and a Salmonella typhimurium strain on fresh produce. Journal of Food Protection, 72(11), 2413-2417.

Simoes, M., Simoes, L. C., Pereira, M. O., & Vieira, M. J. (2008). Sodium dodecyl sulfate allows the persistence and recovery of biofilms of Pseudomonas fluorescens formed under different hydrodynamic conditions. Biofouling, 24(1), 35-44.

Tiwari, T. S., Ray, P. B., Jost, K. C., Rathod, M. K., Zhang, Y., Brown-Elliott, B. A., Hendricks, K., & Wallace, R. J. (2003). Forty years of disinfectant failure: Outbreak of postinfection Mycobacterium abscessus infection caused by contamination of benzalkonium chloride. Clinical Infectious Diseases, 36(8), 954-962.

Vaerewijck, M., Sabbe, K., Bare, J., Spengler, H.-P., Favoreel, H., & Houf, K. (2012). Assessment of the efficacy of benzalkonium chloride and sodium hy-pochlorite against Acanthamoeba polyphaga and Tetrahymena spp. Journal of Food Protection, 75(3), 541-546.

Venkateswara Rao, J., Gunda, V., Srikanth, K., & Arepalli, S. K. (2007). Acute toxicity bioassay using Paramecium caudatum, a key member to study the effects of monocrotophos on swimming behaviour, morphology and reproduction. Toxicological and Environmental Chemistry, 89(2), 307-317.

Walsh, S. E., Maillard, J.-Y., Catrenich, C. E., Charbonneau, D. L., & Bartolo, R. G. (2003). Activity and mechanisms of action of selected biocidal agents on Gram-positive and negative bacteria. Journal of Applied Microbiology, 94(2), 240-247.

Yoshimatsu, T., & Hiyama, K. (2007). Mechanism of the action of didecyldime-thylammonium chloride (DDAC) against Escherichia coli and morphological changes of the cells. Biocontrol Science, 12(3), 93-99.

Zasekin, D. A., Dimko, R O., & Kovalenko, V. L. (2016). Efektivnist' dezinfek-tantu na osnovi organichnikh kislot ta nanochastinok metaliv shhodo testkultur mikroorganizmiv [Efficiency of disinfectant based on organic acids and nanoparticles of metals in relation to test cultures of microorganisms]. Problems of Zoinengineering and Veterinary Medicine, 30(2), 358-360 (in Ukrainian).

Zazharskyi, V. V., Davydenko, P., Kulishenko, O., Chumak, V., Kryvaya, A., Ba-baruk, A., & Borovik, I. (2018b). Porivnyal'na otsinka bakteritsidnikh vlastivostej dezinfektantiv [Comparative assessment of bactericidal properties of disinfectants]. Bulletin of the Sumy National Agrarian University, 42(1), 273-276 (in Ukrainian).

Zazharskyi, V. V., Fotina, T. I., Berezovsky, A. V., Davydenko, P., Kulishenko, O., Chumak, V., Kryvaya, A., & Borovik, I. (2018a). Vpliv dezinfikuyuchikh zasobiv na kriogenni shtami mikroorganizmiv [Influence of disinfectants on cryogenic strains of microorganisms]. The Journal of the Dnipropetrovsk State Agrarian and Economic University, Veterinary Sciences, 47(1-2), 53-58 (in Ukrainian).

Zhmin'ko, P. G., Kokshariova, N. V., & Dmytrenko, M. P. (2006). Dosvid v skri-ningovyh doslidzhenniyah toksitchnosti likarskih zasobiv [Experience of using different test systems in screening studies on drug toxicity]. Bulletin of Pharmacology and Pharmacy, 4, 21-27 (in Ukrainian).